LC-MS analysis of fecal extracts

The method for LC-MS analysis of fecal extracts was as described in a previous study with slight modification⁵⁸. Fecal samples were suspended in 1 ml of 50% acetonitrile (wt/vol) and extracted by vortexing and sonication for 10 min. The suspension was extracted twice by collecting supernatant after centrifuging at 18,000 × g for 10 min. After passage of the supernatant through a 2-μm filter, the filtrate was transferred to a UPLC vial and subjected to LC-MS analysis. A 5-μl aliquot prepared from the fecal extract was injected into an Acquity ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA) and separated in a BEH C18 column (Waters). The mobile phase used a gradient ranging from water to 95% aqueous ACN containing 0.1% formic acid over a 10-min run. The LC eluent was introduced into a Xevo-G2-S quadrupole time-of-flight mass spectrometer (QTOFMS, Waters) for accurate mass measurement and ion counting in negative-mode. Capillary voltage and cone voltage for electrospray ionization was maintained at −3 kV and −35 V for negative-mode detection. Source temperature and desolvation temperature were set at 120 °C and 350 °C, respectively. Nitrogen was used as both cone gas (50 L/h) and desolvation gas (600 L/h), and argon as collision gas. For accurate mass measurement, the mass spectrometer was calibrated with sodium formate solution with mass-to-charge ratio (m/z) of 50–1,000 and monitored by the intermittent injection of the lock mass leucine enkephalin ([M-H]⁻ = m/z 554.2615) in real time. Mass chromatograms and mass spectral data were acquired and processed by the MassLynx^TM software (Waters) in centroided format. Additional structural information was obtained by tandem MS (MS/MS) fragmentation with collision energies ranging from 15 to 40 eV. The concentrations of bile acids in fecal samples were determined by calculating the ratio between the peak area of individual bile acids and the peak area of internal standard and then fitting with a standard curve using the QuanLynx^TM software (Waters). Morphine sulfate standard was purchased from National Institute of Drug Abuse (NIDA) and M3G standard was purchased from Sigma.